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ABSTRACT
Hydrogen peroxide (H2O2) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H2O2 from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin–horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H2O2 production. The rate of H2O2 production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 μmol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca2+, the production rate was very low.
Production of H2O2 from thyroid cells was also measured by a method which discriminated between H2O2 and superoxide anion (O2 −); in this, diacetyl-deuteroheme-substituted HRP was employed as the trapping agent for both O2 metabolites. The thyroid cells produced H2O2, but not O2 −, when the cells were stimulated by extracellular ATP.
Journal of Endocrinology (1990) 126, 283–287
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ABSTRACT
Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate 125I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0·1–0·3 mU/ml. Addition of ClO4 − to the basal medium inhibited transcellular transport, whilst addition of ClO4 − to the apical medium was much less effective.
Constituents labelled with 125I in the apical medium were analysed. The amount of protein-bound 125I measured by acid precipitation was 3–8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate–polyacrylamide gel electrophoresis revealed that most of the 125I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium.
The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential.
Journal of Endocrinology (1990) 126, 275–281
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In this study we describe a novel mutation of the thyroid peroxidase (TPO) gene that resulted in a total iodide organification defect. TPO activity and thyroxine formation in thyroglobulin in the thyroid gland of the patient were below the limits of detection. However, TPO mRNA was detectable at a similar size and concentration as compared with normal thyroid tissues when measured by Northern blot analysis. Sequence analysis of the TPO gene showed the presence of two mutations, a missense mutation in exon 7 and C insertion in exon 14. These mutations were heterozygous and located in different alleles. The latter mutation has already been reported as one of the mutations of the TPO gene resulting in total iodide organification defect. The former mutation was further analysed by mRNA transfection studies in which mutated mRNA was transfected to CHO-K1 cells by electroporation. The results of transfection studies showed that the cells transfected with mutated mRNA expressed similar size TPO molecules to those of cells transfected with wild-type mRNA but that they lacked TPO activity. The two mutations of the TPO gene resulting in the total iodide organification defect in the patient cosegregated from her parents.
